This invention is directed to methods for the preparation of protected forms of oligonucleotides wherein at least one of the phosphate moieties of the oligonucleotide is protected with a protecting group that is removable by intracellular enzymes. The invention is further directed to methods for preparing such oligonucleotides that contain radioactive labels. The invention also is directed to the preparation of amidite reagents for preparing these oligonucleotides. The methods of the invention can be used to prepare prodrug forms of oligonucleotides and chimeric oligonucleotides that are modified with certain functional groups that are cleavable by intercellular enzymes to release the oligonucleotide from its prodrug form. The oligonucleotides prepared by the methods of the invention can be of any known sequence, preferably one that is complementary to a target strand of a mRNA. The compounds produced by the methods of the invention are useful for therapeutics, diagnostics, and as research reagents.
Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2xe2x80x2-O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. For disease states, classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect may be obtained with minimal side effects. It is therefore a general object of such therapeutic approaches to interfere with or otherwise modulate gene expression, which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides, especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence. Several oligonucleotides are currently undergoing clinical trials for such use. Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action. Several recent reports describe such interactions (see Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et. al., Gene 1990, 89, 203-209).
In addition to such use as both indirect and direct regulators of proteins, oligonucleotides and their analogs also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with gene expression inhibition, diagnostic applications utilize the ability of oligonucleotides and their analogs to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligomeric compounds via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry. PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied. For example, PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling. Commercialization has led to the development of kits which assist non-molecular biology-trained personnel in applying PCR. Oligonucleotides and their analogs, both natural and synthetic, are employed as primers in such PCR technology.
Oligonucleotides and their analogs are also used in other laboratory procedures. Several of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et al., Eds., Current Publications, 1993. Such uses include as synthetic oligonucleotide probes, in screening expression libraries with antibodies and oligomeric compounds, DNA sequencing, in vitro amplification of DNA by the polymerase chain reaction, and in site-directed mutagenesis of cloned DNA. See Book 2 of Molecular Cloning, A Laboratory Manual, supra. See also xe2x80x9cDNA-protein interactions and The Polymerase Chain Reactionxe2x80x9d in Vol. 2 of Current Protocols In Molecular Biology, supra.
Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses. Thus a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities. Such modifications include those designed to increase binding to a target strand (i.e. increase their melting temperatures, Tm, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotides and their analogs, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide.
The complementarity of oligonucleotides has been used for inhibition of a number of cellular targets. Such complementary oligonucleotides are commonly described as being antisense oligonucleotides. Various reviews describing the results of these studies have been published including Progress In Antisense Oligonucleotide Therapeutics, Crooke, S. T. and Bennett, C. F., Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-129. These oligonucleotides have proven to be very powerful research tools and diagnostic agents. Further, certain oligonucleotides that have been shown to be efficacious are currently in human clinical trials.
Antisense therapy involves the use of oligonucleotides having complementary sequences to target RNA or DNA. Upon binding to a target RNA or DNA, an antisense oligonucleotide can selectively inhibit the genetic expression of these nucleic acids or can induce other events such as destruction of a targeted RNA or DNA or activation of gene expression.
Destruction of targeted RNA can be effected by activation of RNase H. RNase H is an endonuclease that cleaves the RNA strand of DNA:RNA duplexes. This enzyme, thought to play a role in DNA replication, has been shown to be capable of cleaving the RNA component of the DNA:RNA duplexes in cell free systems as well as in Xenopus oocytes.
RNase H is very sensitive to structural alterations in antisense oligonucleotides. To activate RNase H, a DNA:RNA structure must be formed. Therefore for an antisense oligonucleotide to activate RNase H, at least a part of the oligonucleotide must be DNA like. To be DNA like requires that the sugars of the nucleotides of the oligonucleotide have a 2xe2x80x2-deoxy structure and the phosphate linkages of the oligonucleotide have negative charges. Chemical modifications of the DNA portion of oligonucleotide at either of these two positions resulted in oligonucleotides that are no longer substrates for RNase H.
However, 2xe2x80x2-deoxy nucleotides have weaker binding affinity to their counterpart ribonucleotides than like ribonucleotides would, i.e., RNA:RNA binding is stronger than DNA:RNA binding, and the presence of the negative charges has been thought to contribute to reduced cellular uptake of the antisense oligonucleotide. Therefore, to circumvent the limitations of DNA like oligonucleotides, chimeric oligonucleotides have been synthesized wherein a DNA like central portion having 2xe2x80x2-deoxy nucleotides and negative charged phosphate linkages is included as the center of a large oligonucleotide that has other types of nucleotides on either side of the DNA like center portion. The center portion must be of a certain size in order to activate RNase H upon binding of the oligonucleotide to a target RNA.
There remains a continuing long-felt need for modified antisense compounds that incorporate chemical modifications for improving characteristics such as compound stability, cellular uptake and detectability, but are also available for regulation of target RNA through each of the known mechanisms of action of antisense compounds. Such regulation of target RNA would be useful for therapeutic purposes both in vivo and ex vivo and, as well as, for diagnostic reagents and as research reagents including reagents for the study of both cellular and in vitro events.
Labeling with radioactive isotopes provides an efficient tool for studying pharmacological properties of antisense oligonucleotides. As with other classes of drug compounds, this novel class of therapeutics requires high sensitivity radiodetection for evaluation of distribution of antisense agents in tissues and assessment of their metabolic fate. Several methods to introduce 35S at the internucleosidic thiophosphate of 3H or 14C at the base moiety of synthetic oligonucleotides have been reported. Among these labels, 14C offers the highest specific activity and the longest half-life. Considering catabolism of nucleic acids, labeling with 14C at the C-2 position of thymidine results in formation of 14CO2 which is cumbersome to trap and analyze. On the other hand, labeling at either the C-4 or C-6 position leads to xcex2-aminoisobutyric acid as the metabolite which is much more convenient to analyze.
There remains a need for methods of preparing labeled antisense oligonucleotides that overcome the foregoing difficulties. The present invention is directed to the foregoing important ends.
The present invention is directed to methods for the preparation of oligonucleotides having at least one bioreversible protecting group that confers enhanced chemical and biophysical properties. The bioreversible protecting groups further lend nuclease resistance to the oligonucleotides. The bioreversible protecting groups are removed in a cell, in the cell cytosol, or in vitro in cytosol extract, by endogenous enzymes. In certain preferred oligonucleotides of the invention the bioreversible protecting groups are designed for cleavage by carboxyesterases to yield unprotected oligonucleotides.
In one aspect of the present invention, methods are provided for the preparation of oligomeric compounds having at least one moiety having the Formula I: 
wherein:
Z is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y1 is O or S;
Y2 is O or S;
Y3 is C(xe2x95x90O) or S;
q is 2 to about 4;
R1 is H, OH, F, or a group of formula R7xe2x80x94(R8)n;
R7 is C3-C20 alkyl, C4-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxy, C2-C20 alkenyloxy, or C2-C20 alkynyloxy;
R8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, poly-alkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (xe2x80x94Oxe2x80x94X3)p, where p is 1 to about 10 and X3 is alkyl having from one to about 10 carbons;
B is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled.
These methods comprise the steps of providing a compound having the Formula II: 
wherein:
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R6 is straight or branched chain alkyl having from 1 to 10 carbons.
Compounds of Formula II are then reacted with compounds having Formula III: 
(wherein R3a is hydrogen; m is 0 to about 50; and R2 is a hydroxyl protecting group, or a linker connected to a solid support, provided that R2 and R3 are not both simultaneously a linker connected to a solid support), thereby forming the oligomeric compound.
In some preferred embodiments, the methods of the invention further comprise oxidizing or sulfurizing the oligomeric compound to form a further compound having Formula III, wherein R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support, and where m is increased by n+1. In further preferred embodiments, the methods of the invention include a capping step, which can be performed prior to or subsequent to oxidation or sulfurization.
In preferred embodiments, the methods of the invention further comprise cleaving the oligomeric compound to produce a compound having the Formula IV: 
In some particularly preferred embodiments, the cleaving step occurs enzymatically, more preferably in vivo.
In some preferred embodiments, the compound of Formula II is formed by reaction of a compound having Formula V: 
with a compound having the Formula VI: 
in the presence of an acid.
In further preferred embodiments, the compound of Formula II is obtained by reaction of a compound having Formula V with a chlorophosphine compound of formula ClP[i-Pr2N]2, followed by reaction with a compound of Formula XX: 
in the presence of an acid.
Also provided in accordance with the present invention are methods for the preparation of a phosphoramidite of Formula II: 
wherein:
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
Z is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y1 is O or S;
Y2 is O or S;
Y3 is C(xe2x95x90O) or S;
q is 2 to about 4;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R1 is H, OH, F, or a group of formula R7xe2x80x94(R8)n;
R7 is C3-C20 alkyl, C4-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxy, C2-C20 alkenyloxy, or C2-C20 alkynyloxy;
R8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (xe2x80x94Oxe2x80x94X3)p, where p is 1 to about 10 and X3 is alkyl having from one to about 10 carbons;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R6 is straight or branched chain alkyl having from 1 to 10 carbons.
Such methods comprise the steps of providing a compound Formula V: 
and reacting the compound with a diaminohalophosphine of Formula: 
(wherein X is halogen, with chlorine being preferred), thereby producing a phosphordiamidite of Formula: 
The phosphorordiamidite is then contacted with a regent of Formula XX: 
to produce the phosphoramidite.
Also provided in accordance with the present invention are methods for the preparation of a phosphoramidite of Formula II: 
wherein:
R3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
z is alkyl having from one to about six carbon atoms;
Y1 is O or S;
Y2 is O or S;
Y3 is O or S;
q is 2 to about 4;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R1 is H, OH, F, or a group of formula R7xe2x80x94(R8)n;
R7 is C3-C20 alkyl, C4-C20 alkenyl, C2-C20 alkynyl, C1-C20 alkoxy, C2-C20 alkenyloxy, or C2-C20 alkynyloxy;
R8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (xe2x80x94Oxe2x80x94X3)p, where p is 1 to about 10 and X3 is alkyl having from one to about 10 carbons;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R6 is straight or branched chain alkyl having from 1 to 10 carbons.
These methods comprise the steps of providing a compound Formula V: 
and reacting the compound with a compound of Formula: 
wherein:
Z is alkyl having from one to about six carbon atoms;
Y1 is O or S;
Y2 is O or S;
Y3 is C(xe2x95x90O) or S;
q is 2 to about 4;
R5 is xe2x80x94N(R6)2, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R6 is straight or branched chain alkyl having from 1 to 10 carbons;
thereby producing the phosphordiamidite.
In some preferred embodiments, the compound of Formula: 
is formed by the reaction of a compound of Formula: 
with a compound of Formula: 
in the presence of an acid.
In preferred embodiments of the foregoing methods, Z is methyl, t-butyl or phenyl, with t-butyl being preferred. In some particularly preferred embodiments, n is 0.
In some preferred embodiments of the foregoing methods, R2 is a linker to a solid support.
In preferred embodiments of the foregoing methods, Y1 and Y2 are each O and Y3 is C(xe2x95x90O), or Y1 and Y2 are each S and Y3 is C(xe2x95x90O), or Y1 is S and Y2 is O and Y3 is C(xe2x95x90O). In especially preferred embodiments, Y1 is O and Y2 is S and Y3 is C(xe2x95x90O).
In some preferred embodiments of the foregoing methods, each R6 is isopropyl. In some especially preferred embodiments, n is 0; R3 is H, R5 is diisopropylamino; Y1 is O; Y2 is S; Y3 is C(xe2x95x90O); and Z is methyl, phenyl or t-butyl, with t-butyl being preferred.
In some preferred embodiments of the foregoing methods, B radiolabeled nucleobase. In more preferred embodiments, the radiolabeled nucleobase has the formula: 
wherein denotes a 14C atom.
In preferred embodiments of the foregoing methods, M is an optionally protected phosphite, phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, or alkyl phosphonate internucleotide linkage. In especially preferred embodiments, M is a phosphite, phosphodiester, phosphotriester, phosphorothioate, phosphorodithioate, or alkyl phosphonate internucleotide linkage protected with protecting group of formula xe2x80x94Y1xe2x80x94(CH2)qxe2x80x94Y2xe2x80x94Y3xe2x80x94Z.